This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The human sweet receptor, composed of the monomers T1R2 + T1R3, appears to be the main (and perhaps the only) receptor required to explain sweet taste in humans. When co-expressed with a reporter G-protein in heterologous systems, this heterodimeric receptor responds to the full range of sweet-tasting compounds sensed by humans at concentrations that humans taste. The sweet receptor responds to a surprisingly diverse set of ligands, from small amino acids to moderately sized sweet-tasting plant proteins. No common structure accounts for the sweetness of all of these compounds. Studies from our lab and others indicate that the sweet receptor can be activated by means of a variety of domains and distinct binding sites on the receptor. We have used this diversity in sweet receptor activity as a tool for understanding ligand - receptor interactions as well as for probing the molecular events that lead to activation of this complex receptor. By using heterologous expression, calcium imaging, BRET and mutagenesis and computational modeling in my laboratory and those of my colleagues, we have mapped sweetener binding to at least three domains of the sweet receptor: the venus fly trap module (VFTM) of hT1R2 (various small molecule artificial sweeteners, natural sugars and dipeptide sweeteners), the cysteine-rich domain (CRD) of hT1R3 (brazzein), and the transmembrane domain (TMD) of hT1R3 (cyclamate and NHDC). To date, no sweeteners have been shown to bind in the TMD of T1R2, however, our recent finding suggests that this domain is able to allosterically regulate ligand-induced activity in the sweet receptor. In this proposal, we propose to further determine the characteristics of the hT1R2 TMD that promotes allosteric interactions with other domains of the receptor. We will also determine whether any sweeteners map to its putative intra-helical TMD binding site. In addition to our established techniques (heterologous expression of receptors, mutagenesis, functional assay and computational modeling) for exploring sweetener interactions with the sweet receptor, our collaborator, Fariba Assadi-Porter will use saturation transfer difference (STD) NMR to track ligand-binding to cells expressing full length T1R2 + T1R3 together, each monomer by itself, mutants receptors or parent cells not expressing receptors. This exciting new development will allow us to monitor ligand binding separate from receptor activity. It will also allow us to determine and identify the critical ligand-receptor binding sites that determine sensitivity and selectivity for ligands, in addition to the effect of sweet receptor mutations on the ligand-binding pocket environment(s) by monitoring changes in the NMR spectra for each ligand.